Cancer cells differ from their normal cellular counterparts in many important characteristics, including loss of differentiation, increased genomic instability, and decreased drug sensitivity. Not surprisingly, genetic alterations occur in most, if not all cancer cells, and are thought to lie at the heart of these phenotypic alterations. Furthermore, genetic instability is thought to be required to generate the multiple genetic changes that occur in cancer cells. My laboratory uses somatic cell and molecular genetics to identify and characterize genetic alterations found in tumor cells that induce abnormal cellular phenotypes. By utilizing this approach, my lab has identified a previously unknown chromosomal abnormality that is associated with certain chromosomal rearrangements. This chromosomal phenotype is characterized by a delay in mitotic chromosome condensation, a delay in the chromosome replication timing, and significant chromosomal instability. Chromosomes with this phenotype are common in tumor derived cell lines and in primary tumors. Furthermore, we have found that DNA damage generates chromosomes with this phenotype. Our findings support a model in which the chromosomal instability found in tumor cells, and in cells with DNA damage, stems from a defect in the replication timing of certain chromosomal rearrangements. The experiments described in this proposal are designed to determine the genetic mechanisms responsible for this abnormal chromosomal phenotype. In Specific Aim 1, we will characterize the chromosomal rearrangements generated by ionizing radiation that are associated with delayed replication timing and delayed mitotic condensation. In Specific Aims 2 and 3, we will use chromosome engineering strategies, combined with somatic cell and molecular genetics, to generate specific chromosome deletions and rearrangements that display this abnormal chromosomal phenotype. The long-term goal of these studies is to define the molecular mechanisms responsible for chromosomal instability, one of the most common types of genetic instabilities found in cancer cells.